Development of an amylolytic Lactobacillus plantarum silage strain expressing the Lactobacillus amylovorus α-amylase gene

Abstract

An amylolytic Lactobacillus plantarum silage strain with the starch- degrading ability displayed by Lactobacillus amylovorus was developed. An active fragment of the gene coding for α-amylase production in L. amylovorus was cloned and integrated into the chromosome of the competitive inoculant strain L. plantarum Lp80 at the cbh locus. The α-amylase gene fragment was also introduced into L. plantarum Lp80 on an autoreplicative plasmid. Both constructions were also performed in the laboratory strain L. plantarum NCIB8826. All four recombinant strains secreted levels of amylase ranging from 23 to 69 U/liter, compared with 47 U/liter for L. amylovorus. Secretion levels were higher in L. plantarum NCIB8826 than in L. plantarum Lp80 derivatives and were higher in recombinant strains containing autoreplicative plasmids than in the corresponding integrants. The L. plantarum Lp80 derivative containing the L. amylovorus α-amylase gene fragment integrated into the host chromosome secreted α-amylase to a level comparable to that of L. amylovorus and was stable over 50 generations of growth under nonselective conditions. It grew to a higher cell density than either the parent strain or L. amylovorus in MRS medium containing a mixture of starch and glucose as the fermentable carbohydrate source. This recombinant α-amylolytic L. plantarum strain would therefore seem to have considerable potential as a silage inoculant for crops such as alfalfa, in which water-soluble carbohydrate levels are frequently low but starch is present as an alternative carbohydrate source.